Automated DNA Extraction Research Articles

Comparison of different diagnostic protocols for the detection of Toxocara spp. in faecal samples of cats and dogs

BackgroundToxocara canis and Toxocara cati are parasitic nematodes that occur worldwide. As embryonated Toxocara spp. eggs in the environment pose a zoonotic risk, especially for children, optimal diagnostic approaches are necessary for effective disease response and management, including surveillance. However, little is known about the performance of different diagnostic protocols for detecting Toxocara spp. in the faeces of cats and dogs, hampering movement towards an optimal diagnostic process. This study aimed to compare detection methods, including a newly developed sequential sieving protocol (SF-SSV) and a high-throughput multiplex qPCR-based method to facilitate epidemiological studies.MethodsSpecies-specific Toxocara spp. egg suspensions and canine and feline faecal samples from the field were used to estimate analytical and diagnostic sensitivity of the protocols. The performance of two automated DNA extraction protocols using enzymatic and mechanical lysis were compared by multiplex qPCR, targeting both T. canis and T. cati-specific genomic sequences. All samples were examined by microscopy-based techniques, the sedimentation flotation technique (SF) and a newly developed SF-SSV for the detection, enrichment and purification of parasite eggs. The costs and processing times necessary for all protocols were estimated and compared for both single samples and sets of 100 samples.ResultsTo detect Toxocara spp. eggs, SF-SSV showed the highest analytical sensitivity and a significantly higher diagnostic sensitivity than the DNA detection methods. Mechanical lysis performed better than enzymatic lysis for automated DNA extraction. In automated DNA extraction, 96-well plates performed better than 24-well plates. DNA detection and microscopy-based parasitological methods showed substantial agreement between the results generated by each method. Microscopy-based techniques required the lowest costs and least hands-on time for a single sample. However, when costs and labour were estimated for a set of 100 samples, the DNA detection protocol using 96-well plates for extraction revealed costs similar to SF-SSV and the fastest processing times.ConclusionsSF-SSV was superior in terms of analytical and diagnostic sensitivity for the detection of Toxocara spp. eggs. For larger sets of samples, multiplex qPCR-based DNA detection represents an alternative to microscopy-based methods, based on the possibility of faster sample processing at similar costs to SF-SSV, and the ability to provide species-specific diagnoses.Graphical

Extraction of Genomic DNA from Soil Samples by Polyethylene Glycol-Modified Magnetic Particles via Isopropanol Promotion and Ca2+ Mediation.

Obtaining reliable and informative DNA data from soil samples is challenging due to the presence of interfering substances and typically low DNA yields. In this work, we prepared poly(ethylene glycol)-modified magnetic particles (PEG@Fe3O4) for DNA purification. The particles leverage the facilitative effect of calcium ions (Ca2+), which act as bridges between DNA and PEG@Fe3O4 by coordinating with the phosphate groups of DNA and the hydroxyl groups on the particles. The addition of 2-propanol further enhances this Ca2+-mediated DNA adsorption by inducing a conformational change from the B-form to the more compact A-form of DNA. PEG@Fe3O4 demonstrates a DNA adsorption capacity of 144.6 mg g-1. When applied to the extraction of genomic DNA from soil samples, PEG@Fe3O4 outperforms commercial kits and traditional phenol-chloroform extraction methods in terms of DNA yield and purity. Furthermore, we developed a 16-channel automated DNA extraction device to streamline the process and reduce the extraction time. The successful amplification of target bacterial and fungal amplicons underscores the potential of this automated, device-assisted method for studying soil microbial diversity.

From Jane Doe to Sofia: DNA Extraction Protocol from Bones and Teeth without Liquid Nitrogen for Identifying Skeletal Remains.

DNA analysis plays a crucial role in forensic investigations, helping in criminal cases, missing persons inquiries, and archaeological research. This study focuses on the DNA concentration in different skeletal elements to improve human identification efforts. Ten cases of unidentified skeletal remains brought to the Institute of Forensic Medicine in Timisoara, Romania, underwent DNA analysis between 2019 and 2023. The results showed that teeth are the best source for DNA extraction as they contain the highest concentration of genetic material, at 3.68 ng/µL, compared to the petrous temporal bone (0.936 ng/µL) and femur bone (0.633 ng/µL). These findings highlight the significance of teeth in forensic contexts due to their abundant genetic material. Combining anthropological examination with DNA analysis enhances the understanding and precision of identifying human skeletal remains, thus advancing forensic science. Selecting specific skeletal elements, such as the cochlea or teeth, emerges as crucial for reliable genetic analyses, emphasizing the importance of careful consideration in forensic identification procedures. Our study concludes that automated DNA extraction protocols without liquid nitrogen represent a significant advancement in DNA extraction technology, providing a faster, more efficient, and less labor-intensive method for extracting high-quality DNA from damaged bone and tooth samples.

Validation process of automatic DNA extraction from bone material using a new advanced protocol for the EZ2 Connect instrument

Identification of human remains using genetic methods is an important task of forensic science. DNA markers are proving essential in the identification of unknown human remains. However, environmental factors can lead to poor preservation of DNA, including in bone material. The aim of this study was therefore to compare two methods of DNA isolation from bone material: the traditional organic method and the new protocol using the EZ2 Connect instrument. The study involved three types of bone material, namely molars/premolars, petrous parts of the temporal bone and femurs, all with an estimated PMI of 70–80 years. Importantly, the biological material was obtained from three different environments, categorized as preserving, neutral and degrading, based on basic physico-chemical tests and the potential impact on the bone.The results obtained show that the DNA was best preserved in the petrous bone, followed by the teeth, and the femur. DNA extraction using the EZ2 Connect instrument with a new protocol gave slightly better results for the petrous bone, comparable results for the teeth and worse results for the femur compared to the organic method. Several protocol modifications were tested and optimal conditions for DNA isolation were proposed for the EZ2 protocol. Furthermore, the use of an automated method facilitated the effective accumulation of isolates and increased the chances of successful identification of unknown human remains.

Comparative Study of Two Semi-automated Forensic DNA Extraction Methods

Automation in forensic DNA analysis is crucial for analysts to reduce time, improve results, and decrease risk of contamination. With the variety of commercially available automated DNA extraction systems, comes the need for end-users to be informed of what they provide and what they might lack. Thus, this study aimed to evaluate the efficiency of two semi-automated DNA extraction systems used for forensic DNA analysis: Automate Express™ and Hamilton Microlab STAR™ system, for four parameters; reproducibility, stability, sensitivity and contamination. Overall, the results indicated that both semi-automated systems performed similarly in providing robust and reproducible DNA results while maintaining good capability to overcome PCR inhibition with low risk of contamination. The two semi-automated systems showed higher DNA recovery than organic extraction using phenol-chloroform by 22% for semen and 7% for blood samples. In addition, three sample types, blood, saliva, semen were tested to compare the two systems (total samples n=100). Overall, the data showed the average DNA recovery for Hamilton was higher than the DNA recovery by Automate Express™ for the blood and semen sample types indicating better performance of the Hamilton Microlab STAR™ in terms of recovery and sensitivity level.

Automated DNA extraction from recent and archaeological human skeletal remains using QIAGEN's EZ2 Connect Fx Instrument – an experience report

The aim of this study was to investigate the efficiency of the automated DNA extraction method using the EZ2 Connect Fx instrument (QIAGEN), showcasing its ability to achieve swift and reliable results from bone samples. Genomic DNA extraction from 16 archaelogical and recent bone samples was performed with a commercial EZ1&2® DNA Investigator® Kit (QIAGEN). Prior to PCR amplification, DNA concentration was quantified using Qubit™ Fluorometer (Thermo Fisher Scientific). Amplification was carried out with the Investigator 24plex Kit (QIAGEN). DNA profiles were generated using 3500 Genetic Analyzer (Applied Biosystems) and analysed with GeneMapper™ ID-X 1.6 Software (Applied Biosystems). For nine archaeological dental samples, eight exhibited partial profiles, and one showed no amplified loci. Among the seven recent femoral bone samples, one exhibited a partial profile, while six presented complete DNA profiles. In this study, we investigated the benefits of automated DNA extraction, particularly its ability to generate timely and reliable results. Additionally, this method reduces pipette and tip usage, almost completely minimising the risk of human error, exogenous DNA contamination and cross-contamination. Such efficiency is of paramount importance, especially when working with skeletal remains, namely archaeological skeletal remains that pose a challenge for molecular genetic analyses.

The Rapid Detection of Salmonella enterica, Listeria monocytogenes, and Staphylococcus aureus via Polymerase Chain Reaction Combined with Magnetic Beads and Capillary Electrophoresis.

Food safety concerns regarding foodborne pathogen contamination have gained global attention due to its significant implications. In this study, we developed a detection system utilizing a PCR array combined with an automated magnetic bead-based system and CE technology to enable the detection of three foodborne pathogens, namely Salmonella enterica, Listeria monocytogenes, and Staphylococcus aureus. The results showed that our developed method could detect these pathogens at concentrations as low as 7.3 × 101, 6.7 × 102, and 6.9 × 102 cfu/mL, respectively, in the broth samples. In chicken samples, the limit of detection for these pathogens was 3.1 × 104, 3.5 × 103, and 3.9 × 102 cfu/g, respectively. The detection of these pathogens was accomplished without the necessity for sample enrichment, and the entire protocols, from sample preparation to amplicon analysis, were completed in approximately 3.5 h. Regarding the impact of the extraction method on detection capability, our study observed that an automated DNA extraction system based on the magnetic bead method demonstrated a 10-fold improvement or, at the very least, yielded similar results compared to the column-based method. These findings demonstrated that our developed model is effective in detecting low levels of these pathogens in the samples analyzed in this study. The PCR-CE method developed in this study may help monitor food safety in the future. It may also be extended to identify other foodborne pathogens across a wide range of food samples.

Ultrafast detection of β-lactamase resistance in Klebsiella pneumoniae from blood culture by nanopore sequencing.

Aim: This study aimed to assess the ultra-fast method using MinION™ sequencing for rapid identification of β-lactamase-producing Klebsiella pneumoniae clinical isolates from positive blood cultures. Methods: Spiked-blood positive blood cultures were extracted using the ultra-fast method and automated DNA extraction for MinION sequencing. Raw reads were analyzed for β-lactamase resistance genes. Multilocus sequence typing and β-lactamase variant characterization were performed after assembly. Results: The ultra-fast method identified clinically relevant β-lactamase resistance genes in less than 1h. Multilocus sequence typing and β-lactamase variant characterization required 3-6 h. Sequencing quality showed no direct correlation with pore number or DNA concentration. Conclusion: Nanopore sequencing, specifically the ultra-fast method, is promising for the rapid diagnosis of bloodstream infections, facilitating timely identification of multidrug-resistant bacteria in clinical samples.

Comparative analyses of DNA extraction methods for whole blood quantification of HCMV DNAemia in patients with hematological diseases: false negative cases in manual method

Human cytomegalovirus (HCMV) DNA quantitation in whole blood (WB) by real-time or quantitative polymerase chain reaction (qPCR) is a highly sensitive and reproducible diagnostic procedure for monitoring HCMV DNAemia (DNAemia is the detection of DNA in samples of plasma, whole blood, isolated peripheral blood leukocytes or in buffy-coat specimens) in patients. We provided a comparative analysis of HCMV DNA extraction performance by two different techniques, one performed by an automated extractor and the other by a manual method. We observed that the automated extraction method allowed HCMV DNA detection in the presence of weak viremia while no differences are observed when the viral load is greater. Therefore, automated DNA extraction is a suitable and recommended protocol not only for early detection of HCMV infection but also for more accurate monitoring of HCMV DNAemia during post-therapy follow-up.

Evaluation of low-cost bone and teeth processing methods for automated DNA extraction

Bone and teeth can often be the only, or the best, biological materials available as DNA sources for the identification of decomposed or skeletal human remains. While these hard tissues are resistant to decay and therefore offer endogenous DNA a certain protection from the environment, their mineral matrices make DNA extraction challenging, a problem compounded by the low DNA content and high degradation levels usually associated with these tissues. To help release DNA from the mineral matrix, they are generally ground into a powder to increase their surface area to the extraction reagents. Furthermore, it is now common to perform a demineralization to dissolve the powder and release DNA that remains trapped. A common pulverization method consists of the use of a cryogenic grinder, which prevents heating of the sample and avoids subjecting the DNA to heat damage, but this equipment is costly. That said, little is known about the effect of lower-cost alternatives to the expensive cryogenic grinder on the quality and quantity of DNA recovered. Thus, this study aimed to evaluate inexpensive grinding methods for their potential for use in a forensic laboratory setting. The methods deemed to be viable options for this purpose were investigated to determine their effects on the quantity and quality of extracted DNA. Bone samples were also subjected to freezing prior to pulverization to assess the possibility that a lower sample temperature could limit heat damage to the DNA during processing. Pig bone and tooth powders were extracted using an original and a modified PrepFiler BTA™ Kit protocol, with the latter featuring an added demineralization step. Out of the methods explored, a coffee grinder and mortar and pestle were determined to be the most suitable for bone and teeth processing in a forensic laboratory setting. No significant differences between the two methods were found regarding the quantity and quality of DNA recovered and the rate of powder recovery, although more powder loss occurred when teeth were crushed with the mortar and pestle. Likewise, freezing samples before grinding did not have a significant impact on DNA quality and quantity. Finally, the original DNA extraction protocol performed significantly better than the modified one for the quantity of DNA recovered from both bone and teeth, while the quality was only superior for the bone.

Maintaining the chain of custody: Anti-contamination measures for trace DNA evidence

Trace DNA evidence has become an essential tool in forensic investigations, but its reliability is dependent on strict adherence to protocols and procedures that ensure the integrity of the evidence is preserved throughout the investigation. Maintaining a clear chain of custody is critical to the admissibility of evidence in court, and its absence can result in the exclusion of evidence or even a mistrial being declared. Contamination is a significant issue when collecting DNA evidence at a crime scene, and anti-contamination measures must be taken to ensure the reliability and accuracy of the evidence. The use of technology, such as electronic chain of custody (eCOC), automated DNA extraction systems, real-time PCR analysis, DNA profiling software, and environmental monitoring systems, can enhance the accuracy and reliability of evidence. Law enforcement agencies and forensic labs must establish strict guidelines for the collection, preservation, and analysis of trace DNA evidence to maintain its integrity and admissibility in court.

A fast and highly efficient automated DNA extraction method from small quantities of bone powder from aged bone samples

An efficient extraction method is important for obtaining high-quality DNA from degraded aged bone samples. An automated full-demineralization method using the EDTA and DNA Investigator Kit (Qiagen) combined with Qiagen’s biorobots was optimized in our laboratory in the past to extract the DNA from 500 mg of aged bone samples. The purpose of this research was to further improve the method with the aim of reducing the required sample material, shortening the extraction time, and achieving higher throughput. To process extremely small samples, the amount of bone powder was reduced to 75 mg, EDTA was replaced with reagents from the Bone DNA Extraction Kit (Promega), and decalcification was shortened from overnight to 2.5 h. Instead of 50 ml tubes, 2 ml tubes were used, which allows higher throughput. The DNA Investigator Kit (Qiagen) and EZ1 Advanced XL biorobot (Qiagen) was used for DNA purification. A comparison between both extraction methods was made on 29 Second World War bones and 22 archaeological bone samples. The differences between both methods were explored by measuring nuclear DNA yield and STR typing success. After cleaning the samples, 500 mg of bone powder was processed using EDTA, and 75 mg of powder from the same bone was processed using the Bone DNA Extraction Kit (Promega). DNA content and DNA degradation were determined using PowerQuant (Promega), and the PowerPlex ESI 17 Fast System (Promega) was used for STR typing. The results showed that the full-demineralization protocol using 500 mg of bone was efficient for Second World War and archaeological samples, and the partial-demineralization protocol using 75 mg of bone powder was only efficient for the Second World War bones. The improved extraction method—for which significantly lower amounts of bone powder can be used, the extraction process is faster, and higher throughput of bone samples is possible—is applicable for genetic identification of relatively well-preserved aged bone samples in routine forensic analyses.

Evaluation of an automated platform for non-invasive single-exon fetal RHD genotyping early in pregnancy.

RhD immunization is still the major cause of hemolytic disease of the fetus and newborn. Fetal RHD genotyping during pregnancy followed by tailored anti-D prophylaxis for pregnant RhD-negative women carrying an RHD-positive fetus to prevent RhD immunization is a well-established practice in many countries. This study aimed to validate a platform for high-throughput, non-invasive, single-exon, fetal RHD genotyping consisting of automated DNA extraction and PCR set-up, and a novel system for electronic data transfer to the real-time PCR instrument. We also investigated the effect of storage conditions of fresh or frozen samples on the outcome of the assay. Blood samples from 261 RhD-negative pregnant women collected in Gothenburg, Sweden, between November 2018 and April 2020 during gestation week 10-14 were either tested as fresh after storage for 0-7 days at room temperature or as thawed plasma samples previously separated and stored for up to 13 months at -80°C. Extraction of cell-free fetal DNA and PCR set-up were performed in a closed automated system. Fetal RHD genotyping was determined by real-time PCR amplification of the RHD gene exon 4. The outcome of RHD genotyping was compared with either the results obtained with serological RhD typing of newborns or with the results of RHD genotyping performed by other laboratories. No difference was observed in genotyping results when using fresh or frozen plasma during short- and long-term storage, revealing high stability of cell-free fetal DNA. The assay has shown high sensitivity (99.37%), specificity (100%), and accuracy (99.62%). These data confirm that the proposed platform for non-invasive, single-exon, RHD genotyping early in pregnancy is accurate and robust. Importantly, we demonstrated the stability of cell-free fetal DNA in fresh and frozen samples after short- and long-term storage.

CASE REPORT OF DOUBLE HETEROZYGOUS PATIENTC WITH SPINAL MUSCULAR ATROPHY

Spinal muscular atrophy is an autosomal-recessive disorder characterized by degeneration of motor neurons in the spinal cord and caused by mutations in the survival motor neuron 1 gene, SMN1. In most patients with SMA, the disease is caused by a homozygous deletion or mutation. However, approximately 5% of patients present as compound heterozygotes in which they have only one deletion of SMN1 and a subtle mutation of the other chromosome. The phenotype in these rare cases is much milder than the “classic” forms of SMA, which brings bigger challenge regarding the diagnosis. To describe a case of SMA compound heterozygote and discuss the challenges regarding the diagnosis. Patient’s blood was taken for genetic analysis for SMN1 gene. Automatic DNA extraction, using MLPA method. We present a female patient on the age of 15 months, with clinical features and genetically confirmed spinal muscular atrophy type 2. The genetic analysis showed double heterozygosity for deletion of SMN1, inherited from her mother, as well as genetic conversion of the SMN1 to SMN2 pseudo gene, inherited from the father. The analysis also showed absence of the SMN1 gene and presence of 3 copies of the SMN2 pseudo gene. Fortunately, at our department we are able to treat SMA patients with oral  (Risdiplam) and intrathecal therapy (Nusinersen), which both have shown particular improvement in these patients. Accurate diagnosis of SMA is challenging for compound heterozygotes, but is crucial now, as treatments and gene therapy have become available. Keywords: SMN1 gene, heterozygous, mutation, deletion

Myostatin (MSTN) Gene Detection in Nisi Chicken Using Real-Time PCR

The myostatin (MSTN) gene provides instructions for making myostatin, which helps control the growth and development of tissue throughout the body and limits muscle growth. Myostatin has been studied in mice, cows, and other animals. However, the MSTN gene has not been studied in the nisi chicken. Further, the lack of information using the real-time polymerase chain reaction (PCR) to detect the MTSN gene is still to be explored. Accordingly, this study aimed to detect the MSTN gene in nisi chickens, one of the local-endemic Indonesian chickens, using real-time PCR. DNA of twenty-five male chickens was extracted using an automatic DNA extraction tool, and the MSTN gene was analyzed by real-time PCR Rotor-Gene 5 Plex. Ct and Tm values were indicators of the successful amplification of the MSTN gene. The results showed that the MSTN gene was detected by Ct 16.00 and Tm 80.00, respectively. The study novelty is based on the nisi chicken as the local-endemic chicken from Indonesia as the study objective and the application of the real-time PCR as a method to detect the MSTN gene. From this study, we concluded that real-time PCR is a powerful method to detect the MSTN gene of the nisi chicken. MSTN gene detection using real-time PCR in nisi chickens is a step forward in molecular biology research to identify genetic diversity that can be one of the genetic markers in species DNA research. The aspect that is novel in this study lies in the use of the real-time PCR method in conducting tests to detect DNA species in nisi chickens. The lack of information in the field of research using this technique makes this research essential to be a source of information and reference in similar studies and one of the genetic markers that can apply to other studies in the field of species diversity. This study uses a qualitative testing method with real-time PCR. The interpretation of the results is based on the Ct and Tm values detected during amplification using real-time PCR, where the target gene detected is the MSTN gene. Ct and Tm values were used to see the success of real-time PCR in detecting target genes. Based on the results of real-time PCR, the MSTN gene was detected at Cy 16.00 and Tm. 80.00. In conclusion, the MSTN gene that was the target in this study was detected, which was characterized by amplification of the target DNA as indicated by the Ct and Tm values in real-time PCR.

Novel application of metagenomics for the strain-level detection of bacterial contaminants within non-sterile industrial products - a retrospective, real-time analysis.

The home and personal care (HPC) industry generally relies on initial cultivation and subsequent biochemical testing for the identification of microorganisms in contaminated products. This process is slow (several days for growth), labour intensive, and misses organisms which fail to revive from the harsh environment of preserved consumer products. Since manufacturing within the HPC industry is high-throughput, the process of identification of microbial contamination could benefit from the multiple cultivation-independent methodologies that have developed for the detection and analysis of microbes. We describe a novel workflow starting with automated DNA extraction directly from a HPC product, and subsequently applying metagenomic methodologies for species and strain-level identification of bacteria. The workflow was validated by application to a historic microbial contamination of a general-purpose cleaner (GPC). A single strain of Pseudomonas oleovorans was detected metagenomically within the product. The metagenome mirrored that of a contaminant isolated in parallel by a traditional cultivation-based approach. Using a dilution series of the incident sample, we also provide evidence to show that the workflow enables detection of contaminant organisms down to 100 CFU/ml of product. To our knowledge, this is the first validated example of metagenomics analysis providing confirmatory evidence of a traditionally isolated contaminant organism, in a HPC product.

Characterization of the genetic polymorphism linked to the β-casein A1/A2 alleles using different molecular and biochemical methods.

The 2 major subvariants of β-casein (A1 and A2), coded by CSN2 gene, have received great interest in the last decade both from the scientific community and the dairy sector due to their influence on milk quality. The consumption of the A1 variant, compared with the A2 variant, has a potential negative effect on human health after its digestion but, at the same time, its presence improves the milk technological properties. The aim of the present study was to compare the best method in terms of time required, costs, and technical engagement for the identification of β-casein A1 and A2 variants (homozygous and heterozygous animals) in milk to offer a reliable service for large-scale screening studies. Two allele-specific PCR procedures, namely RFLP-PCR and amplification refractory mutation system (ARMS-PCR), and one biochemical technique (HPLC) were evaluated and validated through sequencing. Manual and automated DNA extraction protocols from milk somatic cells were also compared. Automated DNA extraction provided better yield and purity. Chromatographic analysis was the most informative and the cheapest method but unsuitable for large-scale studies due to lengthy procedures (45 min per sample). Both allele-specific PCR techniques proved to be fast and reliable for differentiating between A1 and A2 variants but more expensive than HPLC analysis. Specifically, RFLP-PCR was the most expensive and labor-demanding among the evaluated techniques, whereas ARMS-PCR was the fastest while also requiring less technical expertise. Overall, automated extraction of DNA from milk matrix combined with ARMS-PCR is the most suitable technique to provide genetic characterization of the CSN2 gene on a large scale.

Comparison of three DNA extraction methods for three different types of fired and unfired ammunition

When handling ammunition for gun loading, epithelial cells from the hands can become adhered to the metal surface, and this trace is a potential source of DNA. This work aimed to compare the efficiency of three DNA extraction methods from fired cartridge cases from three different types of firearms: a 12-gauge shotgun, a point 40 S&W pistol, and a 7.62 mm rifle. Nine volunteers were involved in this study handling 42 pieces of ammunition overall. The unfired ammunition was handled by a known good donor, and we used this data for comparison. DNA profiling was carried out with EZ1 DNA Investigator Kit for EZ1 Advanced XL automated DNA extraction, QIAmp DNA Investigator kit for a non-automated silica-based membrane column method, and direct lysis protocol for a non-automated in-house one. Samples were collected with 0.5 × 0.5 cm pieces of FTA filter paper moistened with distilled water. Quantiplex Pro RGQ kit and Fusion Powerplex 6C were used for genotyping samples. QIAmp DNA Investigator method resulted in the best number of alleles recovered for both conditions tested, both unfired and fired ammunitions: 77 % vs. 19.3 %, followed by the automated extraction (28.6 % vs. 4.3 %) and lysis protocol (0 % vs. 3.9 %). Degradation data from fired cartridge cases were 27 % for column method, 50 % for lysis protocol, and 87 % for EZ1 kit. Kruskal-Wallis test for mean DNA concentration from these samples returned p < 0.05, and Dunn’s multiple comparison test indicated a significant difference between calibers 0.40 S&W and 12-gauge shotgun from lyses protocol method. We did not detect any other significant differences on the test. The 12-gauge shotgun cartridge cases resulted in a high number of alleles overall (56.8 %). The numerous steps for DNA extraction and purification in the column method may explain its better performance. Although the results obtained indicate that all methods be used for DNA extraction from this type of evidence, the silica-based membrane column method appears to be more efficient.

Optimization and implementation of four duplex quantitative polymerase chain reaction assays for gene doping control in horseracing.

The concern about gene doping has remained high in horseracing and other equestrian competitions. Our laboratory has previously developed a duplex quantitative polymerase chain reaction (qPCR) assay capable of detecting in equine blood the human erythropoietin (hEPO) transgene and equine tubulin α 4a (TUBA4A) gene as an internal control the latter providing quality control over DNA extraction and qPCR. This study aimed to optimize the method for routine testing of regulatory samples. The use of an automated DNA extraction system has increased the sample throughput, consistency of DNA extraction, and recovery of reference materials. The use of reduced concentration of primers and hydrolysis probe for internal control minimized their competition with transgene amplification and improved the assay sensitivity. Spike-in of an exogenous internal control at low concentration for plasma analysis has also been validated. Using the new workflow, four duplex qPCR assays have been developed for the detection of transgenes, namely, hEPO, human growth hormone (hGH), insulin-like growth factor 1 (hIGF-1), and equine EPO (eEPO). The estimated limits of detection (LODs) of each transgene were 2000 copies/mL of blood and 200 copies/mL of plasma. This method could detect the presence of transgene in blood and plasma collected from a horse administered intramuscularly (IM) with recombinant adeno-associated virus (rAAV) carrying the hEPO transgene. A longer detection time was observed in blood than in plasma. The methods have been applied to the screening of over a thousand official racehorse samples since June 2020 for the presence of these transgenes.

Comparison of an automated DNA extraction and 16S rDNA real time PCR/sequencing diagnostic method using optimized reagents with culture during a 15-month study using specimens from sterile body sites

Background16S rDNA-PCR for the identification of a bacterial species is an established method. However, the DNA extraction reagents as well as the PCR reagents may contain residual bacterial DNA, which consequently generates false-positive PCR results. Additionally, previously used methods are frequently time-consuming. Here, we describe the results obtained with a new technology that uses DNA-free reagents for automated DNA extraction and subsequent real time PCR using sterile clinical specimens.ResultsIn total, we compared 803 clinical specimens using real time PCR and culturing. The clinical specimens were mainly of orthopedic origin received at our diagnostic laboratory. In 595 (74.1%) samples, the results were concordant negative, and in 102 (12.7%) the results were concordant positive. A total of 170 (21.2%) clinical specimens were PCR-positive, of which 62 (36.5% from PCR positive, 7.7% in total) gave an additional benefit to the patient since only the PCR result was positive. Many of these 62 positive specimens were strongly positive based on crossingpoint values (54% < Cp 30), and these 62 positive clinical specimens were diagnosed as medically relevant as well. Thirty-eight (4.2%) clinical specimens were culture-positive (25 of them were only enrichment culture positive) but PCR-negative, mainly for S. epidermidis, S. aureus and C. acnes. The turnaround times for negative specimens were 4 hours (automated DNA extraction and real time PCR) and 1 working day for positive specimens (including Sanger sequencing). Melting-curve analysis of SYBR Green-PCR enables the differentiation of specific and unspecific PCR products. Using Ripseq, even mixed infections of 2 bacterial species could be resolved.ConclusionsFor endocarditis cases, the added benefit of PCR is obvious. The crucial innovations of the technology enable timely reporting of explicit reliable results for adequate treatment of patients. Clinical specimens with truly PCR-positive but culture-negative results represent an additional benefit for patients. Very few results at the detection limit still have to be critically examined.